Activation of Split RNA Aptamers: Experiments Demonstrating the

Laboratory Experiment Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Activation of Split RNA Aptamers: Experiments Demonstrating the Enzymatic Synthesis of Short RNAs and Their Assembly As Observed by Fluorescent Response Sameer Sajja,† Morgan Chandler,† Caryn D. Striplin,* and Kirill A. Afonin* Department of Chemistry, University of North Carolina at Charlotte, Charlotte, North Carolina 28223, United States

J. Chem. Educ. Downloaded from pubs.acs.org by DURHAM UNIV on 08/30/18. For personal use only.

S Supporting Information *

ABSTRACT: With the advancement of ribonucleic acid (RNA) research in the field of biochemistry, ensuring that undergraduate students have both the theoretical and practical knowledge of current, cutting-edge experimental techniques is of marked importance. Many current biochemistry experiments demonstrate various basic molecular biological techniques including isolation and quantification of nucleic acids, protein gel electrophoresis, and DNA amplification reactions. While covering a wide base of skills, there is an underutilization of RNA as a pedagogical tool. This multistep laboratory experiment introduces students to the emerging field of RNA nanotechnology and to the RNA assembly and subsequent conditional activations of preprogrammed functionalities such as fluorescence. To achieve this, a sequence of standard molecular techniques such as PCR, DNA purification, RNA transcription and purification, RNA quantification, and self-assembly followed by analysis with electrophoretic mobility shift assays and spectroscopic quantification are carried out in a semester-long curriculum offered to undergraduate and lower-level graduate students following a prerequisite biochemistry lab course. KEYWORDS: General, Biochemistry, Laboratory Instruction, Hands-On Learning, Biotechnology, Electrophoresis, Fluorescence Spectroscopy, Molecular Biology, Nanotechnology, Nucleic Acids/DNA/RNA

E

biology, chemistry, material science, and physics. Because the functionality is reliant on structure, chemistry students can utilize RNA to explore biological applications. The recent introduction of RNA aptamers which can be selected (through systematic evolution of ligands by exponential enrichment, or SELEX) to bind specific small molecules for their further fluorescence activation7,8 holds promise for an optimized system which does not require advanced instruments for prompt analysis and imaging.9 Gaining experience in carrying out biochemical experiments within an RNA paradigm not only enhances the skills of undergraduate students and lower-division graduate students to prepare them for careers in science but also benefits the scientific community by opening up the “RNA world” beyond RNA’s intermediate role in the central dogma of molecular biology (DNA → RNA → protein). Designed to build upon a prerequisite biochemistry lab course, this research-focused lab serves as an advanced course and liaison between foundational knowledge covered in chemistry undergraduate programs and its applications in the field. The novel curriculum utilizes the structure−activity relationship of RNA molecules for students

ngagement in laboratory research is an essential component of higher education which provides students with the chance to explore science through investigation rather than static texts. In recent years, there has been a shift in the aims of scientific education toward developing critical thinking skills as well as a deeper understanding for better conceptual applications.1 A combination of project-based and expository laboratory experiments in biochemistry has since been used to introduce a comprehensive array of techniques representative of what is currently being done in both academia and industry.2 Although many such approaches feature one biomoecule for cohesiveness of the teaching lab, only a few focus solely on RNA and its unique functionalities.2,3 With the study of noncoding RNAs as part of the human genome project, it has become apparent that RNA has a multitude of different functions not previously anticipated. This has been the impetus for continued RNA research worldwide.4 Although RNA is well-known for its role as a messenger molecule that relays the genetic information encoded in deoxyribonucleic acid (DNA) for protein expression, its dynamicity in function (i.e., as ribozymes or as regulators of gene expression and editing), programmability, and biocompatibility has led to rapid advances in the field of RNA nanotechnology, which aims to develop RNA-based nanoparticles for a variety of diverse biological applications.5,6 As an educational tool, RNA is a powerful building block bridging the disciplines of © XXXX American Chemical Society and Division of Chemical Education, Inc.

Received: October 4, 2017 Revised: May 15, 2018

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DOI: 10.1021/acs.jchemed.7b00759 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Laboratory Experiment

to achieve the following learning goals: (1) an understanding of innovative research in RNA nanotechnology and how rationally designed RNA nanostructures can be programmed to carry and activate certain functions; (2) experience with nucleicacid-focused biochemical lab techniques (e.g., polymerase chain reaction (PCR), various gel electrophoresis techniques, in vitro transcription, RNA purification and recovery, UV−vis and fluorescent spectroscopies, etc.); (3) the ability to critically analyze, interpret, and present scientific results through the scientific publication process. After the completion of this project, students have developed a better understanding of RNA folding and self-assembly in relation to activity through its interactions with other classes of molecules.

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DEVELOPMENT AND BACKGROUND

RNA Imaging with GFP

The study of RNA has become one of the most important areas in modern biology and medicine. In order to explore the functions of RNA in various cellular systems, RNA imaging is used to visualize the pathways of RNA within a cell. While many traditional assays can provide information about a single time point of cellular RNA interactions, real-time imaging allows for dynamic spatial relationships to be observed.10 While custom fluorophores can be covalently linked to the exogenous synthetic RNAs, one of the most commonly used imaging techniques for intracellular RNAs involves green fluorescent protein (GFP) genetically fused with RNA binding protein (RBP) MS2, so-called the MS2-GFP system.11,12 This imaging technique uses two essential components: (1) RBP from the MS2 bacteriophage which has been fused to GFP and (2) MS2 protein-binding RNA sequences added to the target intracellular RNA. The MS2-GFP system allows for the use of fluorescently tagged programmed RNA sequences in cells. However, unbound GFP-MS2 molecules still fluoresce and thus can cause background noise in the fluorescent signal,12 so the use of multiple GFPs per single RNA has been developed together with variations of this technique such as different tagged RNA sequences and other fluorescent proteins.13,14 One key variant of this technique is the use of a split GFP system.15 The split GFP system utilizes GFP separated into two halves, each of which is fused to a half of an RBP. When the RBPs interact with their target RNA sequences positioned next to each other, the two GFP halves are brought within binding proximity, thus enabling the formation of a complete GFP with restored fluorescent activity.15 Though this system is used to reduce false positives in fluorescence localization, the formed complex still produces background fluorescence.16

Figure 1. F30 Broccoli aptamer and reassociated split F30 Broccoli aptamer (112 nts) bind with DFHBI-1T to induce a fluorescent response, whereas individual split subunits do not. Shown in brackets are the tertiary structure of the F30 Broccoli aptamer (top) compared to the tertiary structure of the assembled split F30 Broccoli aptamer, in which Broc (45 nts) is shown in green and Coli (72 nts) is shown in red (bottom). Structures were created with NUPACK.29,30

selective evolution such as higher thermostability, high expression level, and having a shorter and more stable sequence.17−21 In addition, the attached F30 sequence serves as a scaffold which enhances the fluorescence of Broccoli.21 By splitting the F30 Broccoli aptamer into two separate strands which are inactive when separate but bind the small dye DFHBI-1T upon reassociation, Halman et al. demonstrated an optimized aptameric tool for visualizing interdependent interactions between nucleic acid nanoassemblies.22 Several groups have already employed different split or destabilized aptamers for utilities in biosensing,23,24 tracking the assemblies of RNA nanoparticles25 and the conditional activation of split functionalities.26−28 F30 Broccoli as a Pedagogical Tool

As an optimized and well-established fluorescence imaging tool, the RNA aptamer F30 Broccoli is an exceptionally useful system to introduce students to cutting edge concepts in nanobiotechnology. Therapeutically relevant RNA nanostructures are designed using natural RNA motifs (specific arrangements of secondary structure elements) such as loop receptor interactions, kissing loops, and the phi29 3-way junction, to name a few.5,31,32 RNA nanostructures have the potential to be cotranscriptionally assembled in vitro25,32−34 and thus can also potentially be transcribed and assembled in cells. One way to confirm the correct assembly of RNA nanostructures is through the activation of split fluorescent aptamers embedded into their structure. This approach has been demonstrated in vitro using the split malachite green aptamer.23 Malachite green, however, is known to be toxic to cells and generates large background noise due to nonspecific binding to cellular compartments, whereas the F30 Broccoli aptamer was demonstrated to be active in cells, nontoxic, and with low background noise.21 Using F30 Broccoli to emphasize routine laboratory techniques such as PCR, transcription, gel electrophoresis, and UV−vis and fluorescent spectroscopy allows students to investigate programmable RNA assemblies as well as explore how a diverse set of biochemical concepts are linked together through experimental procedures. By building upon a previously acquired skillset of experimental techniques,

RNA Aptamers as GFP Mimics

To eliminate some of the complexities of the GFP-based system for imaging, a novel RNA mimic of GFP called Spinach was selected by the Jaffrey group.7 This system involves a small dye molecule that mimics the GFP fluorophore. This dye exhibits fluorescence only when bound to a specific RNA aptamer. To increase the binding and subsequent fluorescent signal exhibited with the Spinach aptamer, the Jaffrey group fine-tuned the aptamer for the development of Spinach2 and, eventually, a shorter aptamer called Broccoli (schematically shown in Figure 1).8 Though derived from Spinach, Broccoli shows marked optimization for in vivo studies, especially with the comparatively low magnesium dependence compared to Spinach, which is known to interfere with cellular functions. Broccoli’s improved functionality may be a consequence of its B


RNA purification

RNA precipitation

assembly of Broc+Coli aptamer assembly of Broc+Coli aptamer compile reports

compile reports

presentations

4A, 7B

4B, 8A

5A, 8B

5B, 9A

10A

10B

C

a

present results of split aptamer in a 10 min oral presentation in front of your class mates

learn about primer design and amplify template DNAs for Broc, Coli, and Broccoli by preparing reactions in the thermocycler prepare and run a 1.5% agarose gel to verify PCR products; purify amplified DNA templates prepare and run a 1.5% agarose gel to verify purification; make transcription mixtures using purified DNA templates, NTPs, and buffers (T7 RNA polymerase added by instructors prior to lab 4A) terminate transcription by adding DNase; prepare and run a urea gel to purify RNA; isolate samples for elution using UV shadowing precipitate eluted RNA with ethanol and resolvate in water; measure UV absorbance of RNA molecules using a spectrophotometer assemble the Broc+Coli aptamer with controls and in the presence DFHBI-1T; prepare and run a native-PAGE for visualization assemble the Broc+Coli aptamer with controls; prepare and run a native-PAGE for visualization and stain with DFHBI-1T collaborate with fellow researchers to compile all data and interpret results in a written format; submit results to the class “journal” edit final reports based on feedback of reviewers for resubmission

review safety measures, PPE, and cleaning guidelines calibrate pipettes; prepare glassware; review proper practice for keeping records in scientific notebooks prepare stocks of buffers and reagents to be used throughout the semester

student learning objectives

students demonstrate their understanding of the split aptamer assembly by observing a fluorescent response to confirm the procedure was correctly followed students demonstrate their understanding of the split aptamer assembly by observing a fluorescent response to confirm the procedure was correctly followed gain experience in technical writing and a developed understanding of the scientific review process gain experience in technical writing and a developed understanding of the scientific review process students practice oral speaking skills and demonstrate their findings so that they can be understood by a broad audience

tie in the Beer−Lambert law to spectrophotometry measurements

review correct thermocycler preparation and usage while preparing DNA templates for Broc, Coli, and Broccoli learn purification techniques and how to verify them DNA products demonstrate understanding of the roles of reagents required for RNA transcription in vitro practice denaturing gel and visualization techniques

review correct buffer preparation and the role of common biochemical reagents

good lab practice review review proper pipet use and set up notebooks for recordkeeping integrity

student learning outcomes

During weeks 6A−9A, the objectives of weeks 2B−5B were repeated by students independently. bAll experimental protocols concurrent with this schedule are available in the Supporting Information.

9B

2B, 6A

2A

3A, 6B 3B, 7A

procedureb

introduction to lab introduction to techniques preparation of buffers and reagents polymerase chain reaction (PCR) DNA purification transcription initiation

1A 1B

weeka

Table 1. Schedule of Learning Objectives and Outcomes throughout the 10 Week Biochemistry Lab

Journal of Chemical Education Laboratory Experiment




RNA assemblies, the gels are stained with DFHBI-1T and visualized (Figure 3A,B). As an additional possibility, students may also apply relevant advances in the literature to explore the assembly of the split aptamers by quantifying DFHBI-1T fluorescence on a fluorospectrometer (Figure 3C).22,25 The experiment designed here uses the F30 Broccoli aptamer twice: once as an initial expository walkthrough and a second time as a project-based tool to allow students to independently perform the procedures and validate their performance with fluorescence. Students use their own notebooks for details and think through each procedure individually rather than blindly following the procedure as a group. The class analyzed all data and endeavored to understand the physical association of the dye with the assembled aptamer constructs. All work was organized and presented in manuscripts for submission to the class “journal,” which underwent peer review and editing by other members of the class to allow for practice of technical writing as well as a mock review process.

students focus on the experimental design and outcomes using the F30 Broccoli RNA aptamer. The unique setup of this experimental design, although simple in technique, provides insight about scientific careers to STEM students as they gain authentic laboratory experience over the semester-long curriculum (Table 1).

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CURRICULUM DESIGN This procedure was developed for students to assemble the F30 Broccoli RNA aptamer from two individual RNA strands, called here “Broc” and “Coli”. DNA templates with primers for Broc and Coli are ordered from Integrated DNA Technologies Inc. PCR, DNA purification, in vitro transcription, RNA purification, and recovery are then carried out to synthesize Broc and Coli strands, which are then assembled into a functional aptamer and visualized. All experimental steps are schematically summarized in Figure 2. As a control, an original RNA

Hazards

Gloves, UV-protective goggles, and lab coats are mandatory personal protective equipment (PPE) for all parts of this lab and also aid in avoiding RNase contamination of RNA samples. For native-PAGE experiments, TEMED (which may cause respiratory tract irritation if inhaled) is dispensed from the stock solution in a fume hood. Gloves and goggles are a mandatory part of the lab as TEMED may also cause skin or eye irritation if absorbed through skin or if it comes in contact with eyes. Acrylamide-bis(acrylamide) is toxic if ingested or absorbed through the skin. SYBR II green (Figure 3B) is recommended for staining, as ethidium bromide can act as a frameshift mutagen. Only minute quantities of ethidium bromide should be made available to students and only designated pipettes and tips should be used when dispensing from the fume hood. If ethidium bromide is used, it should be handled with care and properly labeled hazardous waste receptacles must be used to dispose of any pipet tips, gels, or other equipment contaminated with it. Electrophoresis uses high voltage sources and equipment must be operated according to all safety recommendations. Proper safety precautions (wearing PPE) must be taken to avoid exposure to UV light during visualization. Additionally, a UV shield can be used for extra protection when handling the UV lamp. Benefits of Approach

Over the design and implementation of the course, three different instructors have noted an improved understanding of RNA nanotechnology and of biochemical techniques among students during experimental progression. Reliable results similar to those with the control gels (Figure 4A) were observed consistently. Instead of simply following the instructions, the vast majority of students (regardless of their prior research lab experience) actively engaged in asking questions and proposing new experiments. Using the published text accompanying the class as well as the resources provided (see Supporting Information for details), this curriculum may easily be adopted for use at other institutions. The typical length of procedures readily fits into 3 h laboratory time blocks and for longer procedures, preparations can be done beforehand and started by instructors. Additionally, the instruments used (thermocycler, heating blocks, centrifuges, gel electrophoresis systems, UV transilluminator, UV spectrophotometer, fluorospectrometer) are common components of most biochemistry laboratories and will not pose a financial burden on institutions. Alternative

Figure 2. Steps in the assembly of the split F30 Broccoli aptamer. All experimental protocols are available in Supporting Information.

F30 Broccoli aptamer is used. All constructs are analyzed by nondenaturing polyacrylamide gel electrophoresis (nativePAGE) stained with ethidium bromide (EtBr) or SYBR II Green. To assess the activation of fluorescence upon the split D




Figure 3. Expected experimental results. (A) Native-PAGE gel visualized under a UV lamp. Staining with DFHBI-1T shows the formation of the aptamer confirmed by fluorescence. (B) Native-PAGE gels showing various concentrations of the F30 Broccoli aptamer visualized with a UV lamp and ChemiDoc MP imaging. Note that in the case of SYBR II Green (more sensitive stain than EtBr) trace amounts of the alternative higher order nonfunctional assemblies are observed. (C) Fluorescence of DFHBI-1T at various concentrations of F30 Broccoli aptamer measured by a fluorospectrometer. In all experiments, DFHBI-1T was used at 10 μM final concentration.

experiment. During the process, students had a shared writing time which allowed for peer discussion about procedural details and outcomes.

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SUMMARY This course-based research experience is an exceptional opportunity for students to actively engage in the scientific process. The setup is designed as an advanced biochemistry course that utilizes the foundations students developed in their prerequisite biochemistry laboratories to actively engage in a research project. Throughout the semester-long set of experiments described, students developed a robust biochemical skillset with which to carry out novel experimental designs, critically analyze data, and interpret results. Creating more opportunities for student research promotes greater scientific literacy and thus opens up avenues for students to continue their education in daily life. The use of RNA as a pedagogical tool leads students to recognize RNA’s utility and diverse functions. The fluorescence response as shown by the binding of the dye DFHBI-1T to the assembled Broc+Coli RNA aptamer allowed for quick determination of whether or not the procedure was carried out successfully. Additionally, running gel electrophoresis to validate periodic stages through the experiments verified progression through the techniques. After completing this course, students met the learning outcomes for a greater developed biochemical foundation for further exploration in science.

Figure 4. Expected experimental results. (A) Broc, Coli, assembled Broc + Coli, and control Broccoli aptamer (all at 5 μM concentrations) visualized on a native-PAGE stained with either EtBr or DFHBI-1T. Slight excess of Coli strand in the assembly (Broc+Coli) results in faint bands visible with EtBr staining but not with DFHBI1T. The differences in migration between F30 Broccoli and Broc +Coli are observed due to the presence of an additional starting sequence in the latter. (B) Fluorescence observed in the presence of DFHBI-1T with all strands mixed at 5 μM concentration. In all experiments, DFHBI-1T was used at 10 μM final concentration.

protocols have been outlined for PCR so that water baths can be used in place of a thermocycler if necessary.

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Course Assessment

Prelab assessments for each stage of the experiments were collected at the beginning of each lab period to ensure that students were familiar with the tasks to be completed. In addition, postlab assessments for each stage were collected at the beginning of the following lab which asked more in-depth questions about hypothetical troubleshooting and problemsolving. During the second phase of experiments in which students independently began synthesizing RNA for the assembly, assessments asked students to interpret their results and evaluate how variables may have affected them. Following the completion of both experimental stages, students assembled all data from throughout the semester for organization into a final report in the format of a research manuscript with appropriate sections, figures, and displayed data. The prepared reports underwent a peer-review process (by the teaching assistants and instructors) analogous to a journal submission in which students were provided with edits, feedback, and additional questions to ensure a thorough interpretation of the

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.7b00759. Detailed experimental protocols, sample assessments such as pre- and postlab questions, possible experimental outcomes, etc. (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Phone: 1-704-687-0685. Fax: 1-704-687-0960. ORCID

Caryn D. Striplin: 0000-0003-1641-8148 Kirill A. Afonin: 0000-0002-6917-3183 E




Author Contributions

(18) Song, W.; Strack, R. L.; Jaffrey, S. R. Imaging bacterial protein expression using genetically encoded RNA sensors. Nat. Methods 2013, 10 (9), 873−5. (19) Strack, R. L.; Song, W.; Jaffrey, S. R. Using Spinach-based sensors for fluorescence imaging of intracellular metabolites and proteins in living bacteria. Nat. Protoc. 2013, 9 (1), 146−55. (20) Song, W.; Strack, R. L.; Svensen, N.; Jaffrey, S. R. Plug-and-play fluorophores extend the spectral properties of Spinach. J. Am. Chem. Soc. 2014, 136 (4), 1198−201. (21) Filonov, G. S.; Kam, C. W.; Song, W.; Jaffrey, S. R. In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies. Chem. Biol. 2015, 22 (5), 649−660. (22) Halman, J. R.; Satterwhite, E.; Roark, B.; Chandler, M.; Viard, M.; Ivanina, A.; Bindewald, E.; Kasprzak, W. K.; Panigaj, M.; Bui, M. N.; Lu, J. S.; Miller, J.; Khisamutdinov, E. F.; Shapiro, B. A.; Dobrovolskaia, M. A.; Afonin, K. A. Functionally-interdependent shape-switching nanoparticles with controllable properties. Nucleic Acids Res. 2017, 45 (4), gkx008. (23) Kolpashchikov, D. M. Binary malachite green aptamer for fluorescent detection of nucleic acids. J. Am. Chem. Soc. 2005, 127 (36), 12442−3. (24) Afonin, K. A.; Danilov, E. O.; Novikova, I. V.; Leontis, N. B. TokenRNA: a new type of sequence-specific, label-free fluorescent biosensor for folded RNA molecules. ChemBioChem 2008, 9 (12), 1902−5. (25) Afonin, K. A.; Bindewald, E.; Yaghoubian, A. J.; Voss, N.; Jacovetty, E.; Shapiro, B. A.; Jaeger, L. In vitro assembly of cubic RNA-based scaffolds designed in silico. Nat. Nanotechnol. 2010, 5 (9), 676−682. (26) Afonin, K. A.; Desai, R.; Viard, M.; Kireeva, M. L.; Bindewald, E.; Case, C. L.; Maciag, A. E.; Kasprzak, W. K.; Kim, T.; Sappe, A.; Stepler, M.; Kewalramani, V. N.; Kashlev, M.; Blumenthal, R.; Shapiro, B. A. Co-transcriptional production of RNA-DNA hybrids for simultaneous release of multiple split functionalities. Nucleic Acids Res. 2014, 42 (3), 2085−2097. (27) Afonin, K. A.; Viard, M.; Martins, A. N.; Lockett, S. J.; Maciag, A. E.; Freed, E. O.; Heldman, E.; Jaeger, L.; Blumenthal, R.; Shapiro, B. A. Activation of different split functionalities on re-association of RNA-DNA hybrids. Nat. Nanotechnol. 2013, 8 (4), 296−304. (28) Rogers, T. A.; Andrews, G. E.; Jaeger, L.; Grabow, W. W. Fluorescent monitoring of RNA assembly and processing using the split-spinach aptamer. ACS Synth. Biol. 2015, 4 (2), 162−6. (29) Zadeh, J. N.; Steenberg, C. D.; Bois, J. S.; Wolfe, B. R.; Pierce, M. B.; Khan, A. R.; Dirks, R. M.; Pierce, N. A. NUPACK: Analysis and design of nucleic acid systems. J. Comput. Chem. 2011, 32 (1), 170−3. (30) Zadeh, J. N.; Wolfe, B. R.; Pierce, N. A. Nucleic acid sequence design via efficient ensemble defect optimization. J. Comput. Chem. 2011, 32 (3), 439−52. (31) Khisamutdinov, E. F.; Bui, M. N.; Jasinski, D.; Zhao, Z.; Cui, Z.; Guo, P. Simple Method for Constructing RNA Triangle, Square, Pentagon by Tuning Interior RNA 3WJ Angle from 60 degrees to 90 degrees or 108 degrees. Methods Mol. Biol. 2015, 1316, 181−93. (32) Afonin, K. A.; Lin, Y. P.; Calkins, E. R.; Jaeger, L. Attenuation of loop-receptor interactions with pseudoknot formation. Nucleic Acids Res. 2012, 40 (5), 2168−80. (33) Afonin, K. A.; Kireeva, M.; Grabow, W. W.; Kashlev, M.; Jaeger, L.; Shapiro, B. A. Co-transcriptional assembly of chemically modified RNA nanoparticles functionalized with siRNAs. Nano Lett. 2012, 12 (10), 5192−5. (34) Afonin, K. A.; Viard, M.; Tedbury, P.; Bindewald, E.; Parlea, L.; Howington, M.; Valdman, M.; Johns-Boehme, A.; Brainerd, C.; Freed, E. O.; Shapiro, B. A. The Use of Minimal RNA Toeholds to Trigger the Activation of Multiple Functionalities. Nano Lett. 2016, 16 (3), 1746−53.

†

S.S. and M.C. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank undergraduate lab members Dylan Dang, Jared Dahl, Allison Tran, Lauren Lee, Beamlak Worku, and Steven Woods for testing all protocols during development, Melina Richardson for testing protocols and assisting in illustrations, and students enrolled in the CHEM 4090/5090: Nanobiochemistry course at the University of North Carolina at Charlotte.

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Activation of Split RNA Aptamers: Experiments Demonstrating the

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